WO2018113781A1

WO2018113781A1 - Anti-pcsk9 antibody and application thereof

Info

Publication number: WO2018113781A1
Application number: PCT/CN2017/118050
Authority: WO
Inventors: 曾竣玮; 克劳兰⋅埃瑞克; 贝尔克⋅乔纳森·P; 缪小牛; 张敏; 博兰⋅奈德赛卡恩; 刘晓林; 俞德超
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2016-12-24
Filing date: 2017-12-22
Publication date: 2018-06-28
Also published as: JP2023002635A; CN108239150A; CN110177810B; AU2017379048B2; EP3560961A4; KR102331021B1; CA3047049A1; US11485795B2; JP7167027B2; BR112019012648A2; CA3047049C; US20200087416A1; KR20190096411A; JP2020506670A; AU2017379048A1; CN110177810A; EP3560961A1

Abstract

The present invention provides an antibody that specifically binds to proprotein convertase subtilisin/kexin type 9 (PCSK9), an antigen-binding fragment of the antibody, and a composition comprising the antibody or the fragment. Also provided are a nucleic acid that encodes the antibody or the fragment, a host cell containing the nucleic acid, and applications of the antibody and the fragment in treatment and diagnosis.

Description

anti-PCSK9 antibody and use thereof

The present invention relates to novel antibodies and antibody fragments which specifically bind to a proprotein convertase subtilisin/kexin type 9 (PCSK9) (hereinafter referred to as PCSK9) and a composition containing the same or an antibody fragment . Furthermore, the invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, and host cells comprising the same, and related uses. Furthermore, the invention relates to the therapeutic and diagnostic use of these antibodies and antibody fragments.

Background technique

Elevated serum cholesterol levels are important risk factors for cardiovascular events. Currently, the cornerstone of cholesterol-lowering therapy is statins, which play an important role in primary prevention and secondary prevention of atherosclerotic cardiovascular disease. However, current lipid-lowering therapies do not meet clinical needs.

Although statins can reduce cardiovascular death, there are certain limitations in statin therapy. First, statins have a maximum reduction in low-density lipoprotein cholesterol (LDL-C) of 40 to 55%, and doubling the dose of statins can only reduce LDL-C by about 6%.

Large-scale studies have shown that statins/combinations do not meet LDL-C treatment goals. In the L-TAP 2 (Lipid Treatment Assessment Project 2) study, 9955 patients with hyperlipidemia from nine countries in the Americas, Europe, and Asia received stable lipid-lowering therapy (75% of them received statin therapy). The overall LDL-C compliance rate is 47-84% (Waters DD et al, Lipid Treatment Assessment Project (L-TAP) 2: a multinational survey to evaluate the proportion of patients achieving low-density lipoprotein cholesterol goals. Circulation. 1): 28-34, 2009). In a comprehensive clinical analysis of multiple statins, statins have a role in reducing cardiovascular events as primary prevention and secondary prevention, but overall only reduce one-third of the events, especially in high-risk groups. Among them, only 27% of events were reduced (Libby P., The forgotten majority: unfinished business in cardiovascular risk reduction. Journal of the American College of Cardiology. 46 (7): 1225-1228, 2005).

At present, various cholesterol-lowering drugs have been marketed or under research, and anti-PCSK9 antibodies have received extensive attention because of their good safety and efficacy.

PCSK9 is a serine protease belonging to the proprotein convertase family. In rodents and humans, PCSK9 is mainly expressed in the liver, followed by the small intestine and kidney. First, a 72kDa PCSK9 precursor protein was synthesized in the rough endoplasmic reticulum. The precursor protein contains a 30 amino acid N-terminal signal peptide, a leader peptide (31-152), a catalytic region (153-425) and C. Cysteine, histidine-rich domain (CHRD) (426-692) (Duff CJ., et al., Antibody-mediated disruption of the interaction between PCSK-9 and the low- Density lipoprotein receptor. The Biochemical Journal. 419(3): 577-584, 2009; Lambert G., et al., Molecular basis of PCSK-9 function. Atherosclerosis. 203(1): 1-7, 2009). After being autocatalyzed by the Gln ₁₅₂ position, the precursor protein is cleaved into a 14kDa leader peptide fragment and a 63kDa mature functional protein (including a catalytic structure and a C-terminal domain), which are non-covalently tightly bound to form a complex. The leader peptide acts as a molecular chaperone of the mature protein, after which the complex leaves the endoplasmic reticulum to reach the Golgi apparatus, and is secreted from the cells into the blood circulation by tyrosine sulfation, acetylation and a series of post-translational modifications in the Golgi apparatus.

The secreted PCSK9 acts mainly to mediate the degradation of low density lipoprotein receptor (LDLR) on the plasma membrane surface of hepatocytes. The catalytic region of PCSK9 contains a site that binds to LDLR and binds to epidermal growth in LDLR structures. Epidermal growth factor-like repeat homology domain-A (EGF-A) forms an LDLR/PCSK9 complex, which is coated with clathrin and then endocytosed into hepatocytes. In the endosomes of hepatocytes, the interaction between LDLR and PCSK9 enhances the formation of a more stable complex due to the acidic environment, ie, the pH decreases, thereby inhibiting the conformational change of LDLR, preventing the dissociation of LDLR and promoting transport. Proteolytic degradation of LDLR/PCSK9 complexes into lysosomes (Lambert G., et al., Molecular basis of PCSK9 function. Atherosclerosis. 203(1): 1-7, 2009; George M, et al., Looking into the crystal ball -upcoming drugs for dyslipidemia. Journal of Cardiovascular Pharmacology and Therapeutics. 20(1): 11-20, 2016). In vivo, PCSK9 acts by interfering with the low-density lipoprotein cholesterol (LDL-C) clearance pathway. When LDLR and LDL-C bind to endocytosis, bound PCSK9 prevents LDLR from detaching from the LDLR/LDL-C complex and will complex The transport of the substance to the lysosomes degrades, so that LDLR cannot be recycled to the surface of the hepatocytes, thereby reducing the amount of LDLR on the surface of the hepatocytes (Lambert G., et al., Molecular basis of PCSK-9 function. Atherosclerosis. 203(1): 1-7, 2009). In addition, immature PCSK9 in the Golgi apparatus can also directly bind to intracellular LDLR and then degrade into lysosomes, preventing LDLR from being expressed on the surface of hepatocytes (Lambert G., et al., Molecular basis of PCSK-9 function. Atherosclerosis. 203 ( 1): 1-7, 2009; Zhang Y., et al., Dysregulation of the low-density lipoprotein receptor pathway is involved in lipid disorder-mediated organ injury. International Journal of Biological Sciences. 12(5): 569-579, 2016). Therefore, PCSK9 can directly act on LDLR through cell surface and intracellular pathways, reducing LDLR expression on the surface of hepatocytes, decreasing LDL-C by hepatocyte reuptake, resulting in decreased LDL-C clearance and circulating LDL-C levels. Elevation; inhibition of PCSK9 can block the binding of plasma PCSK9 to LDLR, thereby preventing endocytosis and degradation of LDLR, increasing the level and quantity of LDLR expression on the cell surface, increasing the reuptake of LDL-C by LDLR, and ultimately reducing circulating LDL-C. Level, to achieve the direct effect of lowering blood fat.

PCSK9 promotes the degradation of other members of the LDLR family, including Very low density lipoprotein receptor (VLDLR), Apolipoprotein E receptor 2 (apoER2), and LDLR-associated protein 1 (LDLR). -related protein 1, LRP1) (Lambert G., et al., Molecular basis of PCSK-9 function. Atherosclerosis. 203(1): 1-7, 2009). Although PCSK9 also binds to the EGF-A domain of VLDLR and apoER2, the degradation pathways are different, and the physiological significance of such degradation has not been found so far (Burke AC., et al. PCSK-9: regulation and target for drug Development for dyslipidemia. Annual Review of Pharmacology and Toxicology. 13(3), 2016). In addition, in addition to maintaining circulating cholesterol homeostasis, recent studies have shown that scavenger receptor CD36 can also interact with PCSK9, suggesting that PCSK9 may play a role in triglyceride metabolism (Burke AC., et al. PCSK-9: regulation and Target for drug development for dyslipidemia. Annual Review of Pharmacology and Toxicology. 13(3), 2016). In summary, PCSK9 has a very close relationship with lipid circulation and metabolism in the body.

Human genetics studies provide strong evidence supporting the role of PCSK9 in regulating LDL-C levels and the incidence of coronary heart disease. Human studies have confirmed that functional gain mutations in the PCSK9 gene are associated with elevated serum LDL-C levels and early coronary heart disease (Abifadel M., et al., Mutations in PCSK-9cause autosomal dominant hypercholesterolemia. Nature Genetics. 34(2): 154- 156, 2003), and loss of function mutations are associated with lower serum LDL-C levels (Cohen JC., et al, Sequence variations in PCSK-9, low LDL, and protection against coronary heart disease. New England Journal of Medicine. 54(12): 1264-1272, 2006). In a 15-year prospective cohort study (ARIC study), LDK-C levels and risk of coronary heart disease were significantly reduced in PCSK9 nonsense mutation carriers (Cohen JC., et al., Sequence variations in PCSK-9, low LDL) , and protection against coronary heart disease. New England Journal of Medicine. 54(12): 1264-1272, 2006). The study included 3,363 black subjects, 2.6% carrying PCSK9 ^142X or PCSK9 ^679X nonsense mutations, a 28% decrease in LDL-C levels and a 88% reduction in coronary heart disease (Cohen JC) compared with those without mutations. , et al, Sequence variations in PCSK-9, low LDL, and protection against coronary heart disease. New England Journal of Medicine. 54(12): 1264-1272, 2006). The study included 9524 white subjects, 3.2% carrying a PCSK9 ^46L nonsense mutation, a 15% decrease in LDL-C levels, and a 47% reduction in the risk of coronary heart disease (Cohen JC., et al., Sequence variations in PCSK-9, low LDL). , and protection against coronary heart disease. New England Journal of Medicine. 54(12): 1264-1272, 2006). In addition, in a woman carrying a PCSK9 complex heterozygous inactivating mutation, PCSK9 was not detected in plasma, and its serum LDL-C level was extremely low (14 mg/dl), but overall health was good and fertility was available. (Zhao Z., et al., Molecular characterization of loss of function mutations in PCSK-9 and identification of a compound heterozygote. American Journal of Human Genetics. 79(3): 514-523, 2006.).

In vivo animal experiments further revealed the mechanism of action of PCSK9. Elevated serum PCSK9 levels in mice result in a decrease in hepatocyte LDLR protein and an increase in serum total cholesterol levels (Lagace TA., et al., Secreted PCSK-9 deletions the number of LDL receptors in hepatocytes and in livers of parabiotic mice. Journal of Clinical Investigation. 116(11): 2995-3005, 2006), in contrast, PCSK9 knockout mice have elevated levels of LDLR protein in hepatocytes (while the level of messenger RNA in LDLR is unchanged), and the corresponding serum total cholesterol levels are reduced by approximately 50% ( Rashid S., et al., Decreased plasma cholesterol and hypersensitivity to statins in mice lacking PCSK-9. Proceedings of the National Academy of Sciences of the United States of America. 102(15): 5374-5379, 2005).

Therefore, there is substantial evidence that PCSK9 plays a role in the regulation of low-density lipoprotein (LDL); expression or up-regulation of PCSK9 is associated with increased plasma LDL cholesterol levels; a corresponding inhibition or deficiency in PCSK9 expression is associated with decreased plasma levels of LDL cholesterol; And a decrease in LDL cholesterol levels is associated with a sequence change in PCSK9; this has been found to confer protection against coronary heart disease (Cohen, 2006 N. Engl. J. Med. 354: 1264-1272).

In clinical trials, it has been found that a decrease in LDL cholesterol levels is directly related to the grade of coronary events (Law et al, 2003 BMJ 326: 1423-1427). In addition, a modest lifetime reduction in plasma LDL cholesterol levels was found to be associated with a substantial decrease in the incidence of coronary events (Cohen, 2006 N. Engl. J. Med. 354: 1264-1272). This is true even in populations with a high prevalence of non-lipid-related cardiovascular risk factors. Therefore, management control of LDL cholesterol levels can be of great benefit.

Based on this, identifying other molecules that can be used to modulate cholesterol levels and block or inhibit or neutralize the activity of PCSK9 would have tremendous benefits. PCSK9 antibodies and their effects in lowering plasma LDL-C are known in the art. Such PCSK9 antibodies and uses thereof are disclosed, for example, in US 2009/0246192, US 2009/0142352, US 2010/0166768, and WO 2010/029513.

As of now, the known PCSK9 monoclonal antibody Alirocumab (Sanofi/Regeneron product, trade name PRALUENT) and Evolocumab (Amgen product, trade name REPATHA) have shown remarkable effects in various types of primary hypercholesterolemia, 2015 Approved by the US FDA for statin-controlled and familial hypercholesteromia (familial hypercholesteromia, including heterozygous and homozygous familial hypercholesteremia, referred to as HeFH and HoFH, respectively) and coronary atherosclerosis A patient with heart disease.

There is still a need to replace PCSK9 antibodies. Specifically, there is a need for a PCSK9 antibody having high affinity with PCSK9, reliable cell line source, good stability, and high efficiency to reduce LDL-C. Still more specifically, there is a need for an alternative PCSK9 antibody that is highly efficient in reducing LDL-C and providing a sustained onset duration (eg, sustained inhibition of LDL-C levels). Such antibodies will also preferably have good physicochemical properties that facilitate development, manufacture or formulation.

Summary of invention

The invention is based in part on a variety of antibodies directed against PCSK9. PCSK9 presents an important and advantageous therapeutic target, and the invention provides antibodies for the treatment and diagnosis of pathological conditions associated with the expression and/or activity of PCSK9. Accordingly, the invention provides compositions, kits, methods and uses for anti-PCSK9 antibodies, anti-PCSK9 antibodies.

In some embodiments, an anti-PCSK9 antibody or antibody fragment (preferably an antigen-binding fragment) that binds to PCSK9 or a fragment thereof, preferably a human PCSK9 protein, is provided.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR), wherein the HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises and is selected from The amino acid sequences of SEQ ID NOS: 1, 7, 8, 9, 10, 11, 12, 13 and 20 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence of 98% or 99% identity or 100% identity, the HCDR2 comprising at least 90%, 91% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 14, 15, 16, 17, and 21. a 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity amino acid sequence consisting of or consisting of, and HCDR3 comprising and selected from SEQ ID NO: 3 Amino acid sequences having amino acid sequences of 18, 19 and 22 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity Or consist of it.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (LCVR), wherein the LCVR comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises and the SEQ ID The amino acid sequence represented by NO: 4 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity or Composition thereof, LCDR2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:5 Or consisting of or consisting of an amino acid sequence of 100% identity, LCDR3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 of the amino acid sequence set forth in SEQ ID NO: Amino acid sequences of %, 98% or 99% identity or 100% identity consist of or consist of.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 And HCDR3 and said LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises at least 90%, 91 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 8, 9, 10, 11, 12, 13 and % or 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequence consisting of, and HCDR2 comprising and selected from SEQ ID NO: 2 The amino acid sequences of 14, 15, 16, 17, and 21 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity Or consisting of an amino acid sequence, and HCDR3 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 18, 19 and An amino acid sequence of %, 97%, 98% or 99% identity or 100% identity consisting of; wherein LCDR1 comprises an amino group as shown in SEQ ID NO: An acid sequence having or consisting of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity, LCDR2 comprises Having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with the amino acid sequence set forth in SEQ ID NO: Or consisting of an amino acid sequence comprising LCDR3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 of the amino acid sequence set forth in SEQ ID NO: An amino acid sequence of % identity or 100% identity consists of or consists of.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR), wherein the HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises ID NO: amino acid sequence of 1, 7, 8, 9, 10, 11, 12, 13 and 20, or consists of the amino acid sequence; HCDR2 comprises SEQ ID NOS: 2, 14, 15, 16, and The amino acid sequence of 21 consists of or consists of the amino acid sequence; HCDR3 comprises or consists of the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 18, 19 and 22.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (LCVR), wherein the LCVR comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises SEQ ID NO The amino acid sequence shown by: 4 or consists of the amino acid sequence; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5; LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 or The amino acid sequence composition.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 And HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 8, 9, 10, 11, 12, 13 and Composition; HCDR2 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 14, 15, 16, 17, and 21; HCDR3 comprises an amino acid selected from the group consisting of SEQ ID NOs: 3, 18, 19 and The sequence consists of or consists of the amino acid sequence; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 5; LCDR3 The amino acid sequence shown by SEQ ID NO: 6 or consists of the amino acid sequence.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20, and HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition, and HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22; wherein LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, and LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: Or consisting of it, LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:7; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 or Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, and HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Composition; HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4; LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: Its composition; and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region HCVR comprising and selected from the group consisting of SEQ ID NO: 23, 25, 26, 27, 28, 29, 30, 31, The amino acid sequence of 32 and 33 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity of the amino acid sequence or composition.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region LCVR comprising at least 90%, 91%, 92%, 93 of the amino acid sequence set forth in SEQ ID NO: Amino acid sequences of %, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity consist of or consist of.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises and is selected from the group consisting of SEQ ID NO The amino acid sequences of 23, 25, 26, 27, 28, 29, 30, 31, 32 and 33 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 An amino acid sequence of % or 99% identity or 100% identity consists of; the light chain variable region LCVR comprises at least 90%, 91%, 92%, 93% of the amino acid sequence set forth in SEQ ID NO: An amino acid sequence of 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity consists of or consists of.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region HCVR comprising a selected from the group consisting of SEQ ID NO: 23, 25, 26, 27, 28, 29, 30, 31, 32 And consist of or consist of the amino acid sequence of 33.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region LCVR comprising or consisting of the amino acid sequence set forth in SEQ ID NO:24.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises a member selected from the group consisting of SEQ ID NO: The amino acid sequence of 23, 25, 26, 27, 28, 29, 30, 31, 32 and 33 consists of or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 23 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 25 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 26 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 27 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 28 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 29 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 30 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 31 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 32 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown in SEQ ID NO: 24.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the heavy chain variable region HCVR comprises SEQ ID NO The amino acid sequence shown by: 33 or consists of; the light chain variable region LCVR comprises or consists of the amino acid sequence shown by SEQ ID NO: 24.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain, wherein the heavy chain comprises and is selected from the group consisting of SEQ ID NO: 34, 36, 37, 38, 39, 40, 41, 42, 43 and The amino acid sequence of 44 has or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity.

In some embodiments, the anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain, wherein the light chain comprises at least 90%, 91%, 92%, 93% of the amino acid sequence set forth in SEQ ID NO: Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity consist of or consist of.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises and is selected from the group consisting of SEQ ID NO: 34, 36, 37, 38, 39, 40, 41, 42 , amino acid sequences of 43 and 44 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity, or Its composition; the light chain comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:35. Or consist of or consist of an amino acid sequence of 100% identity.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain, wherein the heavy chain comprises selected from the group consisting of SEQ ID NOs: 34, 36, 37, 38, 39, 40, 41, 42, 43 and 44 The amino acid sequence consists of or consists of.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain, wherein the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:35.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises a plurality selected from the group consisting of SEQ ID NO: 34, 36, 37, 38, 39, 40, 41, The amino acid sequence of 43 and 44 consists of or consists of; the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In a preferred embodiment, the invention provides an anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44; the light chain comprises SEQ ID NO: The amino acid sequence shown by or consisting of 35.

In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of an anti-PCSK9 antibody, as well as antibodies that bind to the same epitope as any of the antibodies described above.

In certain embodiments, an antibody or antibody fragment (preferably an antigen-binding fragment) that binds to PCSK9 or a fragment thereof is provided, wherein the antibody binds to an epitope within a fragment of PCSK9. In certain embodiments, an antibody or antibody fragment that binds to PCSK9 or a fragment thereof, wherein the antibody binds to a fragment within a fragment of PCSK9 comprising amino acids 75 to 93 and 100 to 110 of human PCSK9 amino acid sequence SEQ ID NO: Bit. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises residue Y78 of human PCSK9. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises the residue T86 of human PCSK9. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises residue H87 of human PCSK9. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises residues Y78, T86 and H87 of human PCSK9. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises residue R104 of human PCSK9. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises residues Y78, T86, H87 and R104 of human PCSK9. In certain embodiments, the functional and/or structural epitope comprises one or more of residues Y78, T86, H87 and R104 of human PCSK9. In certain embodiments, the functional and/or structural epitope comprises one or more of the residues of Y78, T86, H87 and R104 adjacent to human PCSK9. In certain embodiments, the functional and/or structural epitope of an antibody according to the invention comprises (i) at least one residue selected from the group consisting of Y78, T86 and H87 of human PCSK9, (ii) R104 of human PCSK9. In certain embodiments, the functional and/or structural epitope comprises one, two, three or all of the following residues: Y78, T86, H87 and R104 of human PCSK9.

In some embodiments, the anti-PCSK9 antibody is a monoclonal antibody. In some embodiments, the anti-PCSK9 antibody is humanized. In some embodiments, the anti-PCSK9 antibody is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-PCSK9 antibody is a human consensus framework sequence. In one embodiment, an anti-PCSK9 antibody of the invention further encompasses an antibody fragment thereof, preferably an antibody fragment selected from the group consisting of a Fab, Fab'-SH, Fv, scFv or (Fab') ₂ fragment.

In one aspect, the invention provides a nucleic acid encoding any of the above anti-PCSK9 antibodies or fragments thereof. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of a yeast cell, a mammalian cell, or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof. In another embodiment, the host cell is prokaryotic.

In one embodiment, the invention provides a method of making an anti-PCSK9 antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises the step of expressing an antibody or fragment thereof (preferably an antigen-binding fragment) suitable for expression. The host cell is cultured under conditions of a nucleic acid, and the antibody or fragment thereof (preferably an antigen-binding fragment) is optionally isolated. In a certain embodiment, the method further comprises recovering an anti-PCSK9 antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

In some embodiments, the invention provides compositions comprising any of the anti-PCSK9 antibodies or fragments thereof, preferably antigen-binding fragments thereof, described herein, preferably the compositions are pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In one aspect, the invention relates to a method of inhibiting binding of PCSK9 to LDL-receptor (LDLR) in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies described herein or Its fragment. The invention further relates to the use of any of the anti-PCSK9 antibodies or fragments thereof described herein for the preparation of a composition or medicament for inhibiting the binding of PCSK9 to LDL-receptor (LDLR) in a subject.

In another aspect, the invention relates to a method of lowering cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In one embodiment, the cholesterol is LDL-cholesterol, preferably serum cholesterol. In another aspect, the invention relates to a method of lowering LDL-cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In some embodiments, the invention relates to a method of reducing serum LDL-cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In another aspect, the invention relates to any of the anti-PCSK9 antibodies or fragments thereof described herein for use in lowering a subject's cholesterol level (in one embodiment, LDL-cholesterol levels or serum LDL- in a subject) The use of drugs for cholesterol levels).

In another aspect, the invention relates to a method of treating a condition associated with elevated LDL-cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies described herein Or a fragment thereof. The invention further relates to the use of any of the anti-PCSK9 antibodies or fragments thereof described herein for the preparation of a medicament for treating a subject with a condition associated with elevated LDL-cholesterol levels in a subject.

In one aspect, the invention relates to a method of treating a cholesterol-related disease, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. The invention also relates to the use of any of the anti-PCSK9 antibodies or fragments thereof described herein for the manufacture of a medicament for the treatment of a cholesterol-related disease. Exemplary and non-limiting examples of cholesterol related diseases are provided below. In some embodiments, the cholesterol-related disease is hypercholesterolemia or hyperlipidemia. In some embodiments, the invention relates to a method of treating hypercholesterolemia and/or hyperlipidemia, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In some embodiments, the invention further relates to the use of any of the anti-PCSK9 antibodies or fragments thereof described herein for the manufacture of a medicament for the treatment of hypercholesterolemia and/or hyperlipidemia.

In one aspect, the invention relates to a method of treating any disease or condition that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing PCSK9 activity. In some embodiments, a disease or condition that can be treated or prevented by the use of statins can also be treated with any of the anti-PCSK9 antibodies or fragments thereof described herein. In some embodiments, a disease or condition that can benefit from preventing cholesterol synthesis or increased expression of LDLR can also be treated using any of the anti-PCSK9 antibodies or fragments thereof described herein.

In some embodiments, the methods described herein further comprise administering to the subject an effective amount of a second drug, wherein the anti-PCSK9 antibody or fragment thereof described herein is a first drug. In one embodiment, the second agent increases the level of LDLR protein. In another embodiment, the second drug lowers the level of LDL-cholesterol. In another embodiment, the second drug comprises a statin. In another embodiment, the second drug is a statin. In some embodiments, the statin is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, and pravastatin Pravastatin, rosuvastatin, simvastatin, and any combination thereof. In another embodiment, the second drug increases the level of HDL-cholesterol. In some embodiments, the subject or individual is a mammal, preferably a human.

In one aspect, the invention relates to a method of detecting a PCSK9 protein in a sample, the method comprising: (a) contacting the sample with any of the anti-PCSK9 antibodies or fragments thereof described herein; and (b) detecting the anti-PCSK9 antibody or fragment thereof Formation of a complex with PCSK9 protein. In one embodiment, the anti-PCSK9 antibody is detectably labeled.

The invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein, or any combination thereof, are suitable for use in any and all of the anti-PCSK9 antibodies or fragments, methods and uses thereof of the invention described herein.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1. Shows the ability of various anti-PCSK9 antibodies at different concentrations to block the binding of PCSK9 to LDLR.

Figure 2. Shows that varying concentrations of each anti-PCSK9 antibody increases the ability of HepG2 cells to restore LDLR.

Figure 3. Shows the ability of various anti-PCSK9 antibodies at different concentrations to reduce cellular LDLR internalization.

Figure 4. Sequence information showing the FR and CDR of exemplary antibodies of the invention.

Figure 5. Sequence information showing the heavy chain variable region and the light chain variable region of exemplary antibodies of the invention.

Figure 6. HM4High Mass MALDI ToF analysis of complex anti-PCSK9 antibody/PCSK9-WT showing anti-PCSK9 antibody = 0.5 μM; PCSK9-WT = 4 μM, total volume = 10 μl; cross-linking: K200, 180 min incubation time .

Figure 7 shows trypsin, chymotrypsin, Asp-N, elastase and thermolysin peptides of PCSK9-WT. 88.77% of the sequence was covered by the identified peptide.

Figure 8 shows the interaction between PCSK9-WT and anti-PCSK9 antibodies.

Figure 9 is a graph showing the % change rate (average) versus time of LDL-C in serum relative to pre-dose levels (baseline before D1 administration) after subcutaneous or intravenous administration of anti-PCSK-9 antibody or Evolocumab in rats. .

Figure 10 is a graph showing the % change rate (average) vs time of serum % HDL-C relative to pre-dose levels (pre-D1 administration, baseline) after subcutaneous or intravenous administration of anti-PCSK-9 antibody or Evolocumab in rats. Map.

Figure 11 is a graph showing the % change rate (average) versus time of serum LDL-C versus pre-dose levels (baseline before D1 administration) after administration of cynomolgus anti-PCSK-9 antibody or Evolocumab.

Figure 12 is a graph showing the % change rate (average) vs time of serum HDL-C relative to pre-dose levels (baseline before D1 administration) after administration of cynomolgus anti-PCSK-9 antibody or Evolocumab.

Figure 13 is a graph showing the % change rate (average) vs time of serum TC relative to the pre-dose level (baseline before D1 administration) after administration of cynomolgus anti-PCSK-9 antibody or Evolocumab.

Detailed description of the invention

definition

Before the present invention is described in detail below, it is to be understood that the invention is not limited to the particular methodology, aspects, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiment All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined.

For the purpose of interpreting the specification, the following definitions will be used, and the terms used in the singular may also include the plural, and vice versa, as appropriate. It is understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting.

The term "about" when used in connection with a numerical value is meant to encompass a numerical value within the range of the lower limit of 5% less than the specified numerical value and the upper limit of 5% greater than the specified numerical value.

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of a binding pair (eg, an antibody and an antigen), unless otherwise indicated. The affinity of molecule X for its partner Y is generally expressed by the equilibrium dissociation constant (K _D ). Affinity can be measured by conventional methods known in the art, including those known in the art and described herein.

The term "anti-PCSK9 antibody", "anti-PCSK9", "PCSK9 antibody" or "antibody that binds to PCSK9" refers to an antibody which is capable of binding a PCSK9 protein or a fragment thereof with sufficient affinity such that the antibody can be used Targeting diagnostic agents and/or therapeutic agents in PCSK9. In one embodiment, the anti-PCSK9 antibody binds to an unrelated, non-PCSK9 protein to a lesser extent than about 10% of the binding of the antibody to PCSK9, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody against PCSK9 has an equilibrium dissociation constant (K _D ) ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (eg, 10 ^-8 M or less). , for example, 10 ^-8 M to 10 ^-13 M, for example 10 ^-9 M to 10 ^-13 M).

As used herein, "monoclonal antibody" or "mAb" refers to a single copy or cloned antibody derived from, for example, a eukaryote, a prokaryote, or a phage clone, and does not refer to a method of its production. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant techniques, phage display technology, synthetic techniques such as CDR grafting, or a combination of such or other techniques known in the art.

"Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds.

An antibody that "binds to the same epitope as a reference antibody" refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, whereas the reference antibody blocks more than 50% in a competition assay. The binding of the antibody to its antigen.

There are five major classes of antibodies known in the art: IgA, IgD, IgE, IgG and IgM, and several of these antibodies can be further divided into subclasses (isotypes), for example, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

The term "cytotoxic agent" as used in the present invention refers to a substance which inhibits or prevents cell function and/or causes cell death or destruction.

The term "diabodies" refers to an antibody fragment having two antigen binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Variable domain (VH). By using a linker that is too short to be able to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described, for example, in EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA ) 90:6444-6448 (1993). Tri- and tetra-antibodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).

"Effector function" refers to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) Down); and B cell activation.

The term "effective amount" refers to an amount or dose of an antibody or fragment of the invention that, when administered to a patient in single or multiple doses, produces the desired effect in the patient being treated. An effective amount can be readily determined by the attending physician as a person skilled in the art by considering various factors such as the species of the mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease; Response of an individual patient; specific antibody administered; mode of administration; bioavailability characteristics of the administered formulation; selected dosing regimen; and use of any concomitant therapy.

"Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterozygous, chimeric Humanized (especially grafted with CDRs), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab') ₂ fragments, fragments produced by Fab expression libraries, Fd, Fv, II Sulfide-linked Fv (dsFv), single-chain antibody (eg, scFv), diabody or tetra-antibody (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (14), 6444-6448), Nanobody (nanobody) (also known as single domain antibody), anti-idiotypic (anti-Id) antibody (including, for example, an anti-Id antibody against an antibody of the invention), and an epitope-binding fragment of any of the above.

A "Fab" fragment includes a heavy chain variable domain and a light chain variable domain, and also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for a Fab' in which a cysteine residue of a constant domain carries a free thiol group. The F(ab') ₂ antibody fragment was originally produced as a pair of Fab' fragments with a hinge cysteine between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, which is also referred to as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National. Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to a variable domain residue other than a hypervariable region (HVR) (eg, a complementarity determining region) residue. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences typically appear in the following sequences of the heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-H1(L1)-FR2-H2(L2)-FR3 -H3(L3)-FR4.

The terms "full length antibody", "intact antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

"Fv" is the smallest antibody fragment that contains the entire antigen binding site. In one embodiment, the double-stranded Fv species consists of one heavy chain variable domain and one light chain variable domain in a tight, non-covalently associated dimer. In a single-chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can be similar to a double-stranded Fv species. Dimer" structure association. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. In summary, six HVRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although the affinity is lower than the intact binding site. For a review of scFv see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York, 1994), pp. 269-315.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid is introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cell are included herein.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of such an antibody produced by a human or human cell or derived from a non-human source, which utilizes a human antibody library or other human antibody. Coding sequence. This definition of a human antibody specifically excludes a humanized antibody comprising a non-human antigen-binding residue.

"Human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is selected from subtypes of variable domain sequences. In general, the subtype of this sequence is a subtype as in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subtype is subtype kappa I as in Kabat et al. (supra). In one embodiment, for VH, the subtype is subtype III as in Kabat et al. (supra).

A "humanized" antibody refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the HVRs (eg, CDRs) correspond to those of non-human antibodies, and all Or substantially all of the FRs correspond to those of human antibodies. The humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.

The term "hypercholesterolemia" as used herein refers to a condition in which cholesterol levels rise above a desired level. In some embodiments, the LDL-cholesterol level rises above a desired level. In some embodiments, serum LDL-cholesterol levels are elevated above a desired level.

An "immunoconjugate" is an antibody that is conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (eg, cows, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates such as monkeys), rabbits, and rodents (eg, , mice and rats). In some embodiments, the individual or subject is a human.

An "isolated" antibody is one which has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reversed phase) Determined by HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al, J. Chromatogr. B848: 79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that typically comprises the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-PCSK9 antibody or antigen-binding fragment thereof" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or antigen-binding fragments thereof), including in a single vector or separate vectors. Nucleic acid molecules, as well as such nucleic acid molecules present at one or more positions in a host cell.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the alignment of the sequences (and, if necessary, introduction of vacancies) to obtain maximum percent sequence identity, without any conservative substitutions. The percentage of amino acid residues in the candidate sequence to the same amino acid residue in the reference polypeptide sequence after the portion of sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for the alignment, including any algorithms required to obtain the maximum alignment for the full length of the sequences being compared.

When percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. The full length calculation relative to the longer sequence applies to both the nucleic acid sequence and the polypeptide sequence.

The term "pharmaceutical composition" refers to a formulation that is present in a form that permits the biological activity of the active ingredient contained therein to be effective, and does not comprise additional ingredients that have unacceptable toxicity to the subject to which the formulation is administered. .

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient or vehicle with which the therapeutic agent is administered.

Unless otherwise stated, the term "proprotein convertase subtilisin/kexin type 9 (PCSK9)", "PCSK9" or "NARC-1" as used herein refers to from any Any natural PCSK9 of vertebrate origin (including mammals such as primates (eg, humans) and rodents (eg, mice and rats) unless otherwise stated. The term encompasses "full length" unprocessed PCSK9 as well as any form of PCSK9 produced by intracellular processing or any fragment thereof. The term also encompasses variants of naturally occurring PCSK9, for example, splice variants or allelic variants.

The term "PCSK9 activity" or "biological activity" of PCSK9, when used herein, includes any biological effect of PCSK9. In some embodiments, the PCSK9 activity comprises the ability of PCSK9 to interact or bind to a substrate or receptor. In some embodiments, the biological activity of PCSK9 is the ability of PCSK9 to bind to LDL-receptor (LDLR). In some embodiments, PCSK9 binds to and catalyzes a reaction involving LDLR. In some embodiments, PCSK9 activity includes the ability of PCSK9 to reduce or reduce the availability of LDLR. In some embodiments, the biological activity of PCSK9 includes the ability of PCSK9 to increase the amount of LDL in a subject. In some embodiments, the biological activity of PCSK9 comprises the ability of PCSK9 to reduce the amount of LDLR in a subject that can be used to bind to LDL. In some embodiments, the biological activity of PCSK9 comprises the ability of PCSK9 to reduce the amount of LDLR that can be used to bind to LDL. In some embodiments, the biological activity of PCSK9 includes any biological activity resulting from PCSK9 signaling.

As used herein, "treating" refers to slowing, interrupting, arresting, ameliorating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in binding of an antibody to an antigen. The variable domains of the heavy and light chains of a native antibody typically have similar structures, wherein each domain comprises four conserved framework regions (FR) and three complementarity determining regions. (See, eg, Kindt et ^{Kuby Immunology, 6 th ed.,} WHFreeman and page Co.91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies that bind to the antigen can be isolated using a VH or VL domain from an antibody that binds to a particular antigen to separately screen a library of complementary VL or VH domains. See, for example, Portolano et al, J. Immunol. 150: 880-887 (1993); Clarkson et al, Nature 352: 624-628 (1991).

The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes a vector that is a self-replicating nucleic acid structure and a vector that binds to the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "cholesterol-related disease" includes any one or more of the following: hypercholesterolemia, hyperlipidemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, Stroke, cardiovascular diseases, Alzheimers disease, and general dyslipidemia (shown as, for example, increased total serum cholesterol, elevated LDL, increased Triglycerides, increased VLDL and/or low HDL). Some non-limiting examples of primary and secondary dyslipidemia that can be treated with an anti-PCSK9 antibody (alone or in combination with one or more other drugs) include metabolic syndrome, diabetes mellitus, Familial combined hyperlipidemia, familial hypertriglyceridemia, familial hypercholesterolemia, including heterozygous hypercholesterolemia , homozygous hypercholesterolemia, familial defective apoplipoprotein B-100; polygenic hypercholesterolemia; remnant removal disorder (remnant removal) Disease), hepatic lipase deficiency; secondary to any of the following dyslipidemia: dietary indiscretion, hypothyroidism, drugs (including estrogen and progesterone therapy, beta Blockers and thiazide diuretics Nephrotic syndrome, chronic renal failure, Cushing's syndrome, primary biliary cirrhosis, glycogen storage diseases ), hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormone deficiency, and alcohol-induced hypertriglyceridemia Alcohol-induced hypertriglyceridemia.

Antibody of the invention

In one aspect of the invention, provided herein are antibodies against PCSK9, as well as antigen-binding fragments thereof. In some embodiments, the anti-PCSK9 antibody inhibits or blocks PCSK9 activity. In certain embodiments, the antibodies provided herein have an equilibrium dissociation constant (K _D ) ≤ about 1 μM, ≤ about 100 nM, ≤ about 10 nM, ≤ about 1 nM, ≤ about 0.1 nM, ≤ about 0.01 nM, or ≤ about 0.001 nM (eg, 10 ^-8 M or less, such as 10 ^-8 M to 10 ^-13 M, for example, 10 ^-9 M to 10 ^-13 M).

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR), wherein the HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises and is selected from The amino acid sequences of SEQ ID NOS: 1, 7, 8, 9, 10, 11, 12, 13 and 20 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, An amino acid sequence of 98% or 99% identity or 100% identity, the HCDR2 comprising at least 90%, 91% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 14, 15, 16, 17, and 21. a 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity amino acid sequence consisting of or consisting of, and HCDR3 comprising and selected from SEQ ID NO: 3 Amino acid sequences having amino acid sequences of 18, 19 and 22 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity Or consist of it. In certain embodiments, the CDR of the HCVR of the anti-PCSK antibody (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the reference sequence) The amino acid sequence of the CDR of identity or 100% identity comprises one or more substitutions (eg, conservative substitutions), insertions or deletions relative to the corresponding reference sequence, but the anti-PCSK9 antibody comprising the CDRs has the ability to bind PCSK9 .

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (LCVR), wherein the LCVR comprises a complementarity determining region (CDR) LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises and the SEQ ID The amino acid sequence represented by NO: 4 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity or Composition thereof, LCDR2 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence set forth in SEQ ID NO:5 Or consisting of or consisting of an amino acid sequence of 100% identity, LCDR3 comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 of the amino acid sequence set forth in SEQ ID NO: Amino acid sequences of %, 98% or 99% identity or 100% identity consist of or consist of. In certain embodiments, the CDR of the LCVR of the anti-PCSK antibody (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with the reference sequence) The amino acid sequence of the CDR of identity or 100% identity comprises one or more substitutions (eg, conservative substitutions), insertions or deletions relative to the corresponding reference sequence, but the anti-PCSK9 antibody comprising the CDRs has the ability to bind PCSK9 .

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (HCVR), wherein the heavy chain variable region HCVR comprises and is selected from the group consisting of SEQ ID NOs: 23, 25, 26, The amino acid sequences of 28, 29, 30, 31, 32 and 33 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% An amino acid sequence of the same identity or consists of it. In some embodiments, the heavy chain variable region HCVR of an anti-PCSK antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 26, 27, 28, 29, 30, 31, 32, and 33 Or a plurality of (eg, conservatively substituted), inserted or deleted amino acid sequences, but the anti-PCSK9 antibody comprising the HCVR has the ability to bind PCSK9.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region (LCVR), wherein the light chain variable region LCVR comprises at least 90% of the amino acid sequence set forth in SEQ ID NO: The amino acid sequence of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity consists of or consists of. In some embodiments, the light chain variable region LCVR of an anti-PCSK antibody comprises an amino acid sequence having one or more substitutions (eg, conservative substitutions), insertions or deletions as compared to the amino acid sequence set forth in SEQ ID NO:24, However, anti-PCSK9 antibodies comprising the LCVR have the ability to bind PCSK9.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain, wherein the heavy chain comprises and is selected from the group consisting of SEQ ID NO: 34, 36, 37, 38, 39, 40, 41, 42, 43 and The amino acid sequence of 44 has or consists of an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity. In some embodiments, the heavy chain of an anti-PCSK antibody comprises one or more substitutions compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, 37, 38, 39, 40, 41, 42, 43 and 44 (eg, conservative substitution), an inserted or deleted amino acid sequence, but an anti-PCSK9 antibody comprising the heavy chain has the ability to bind PCSK9.

In some embodiments, an anti-PCSK9 antibody or antigen-binding fragment thereof of the invention comprises a light chain, wherein the light chain comprises at least 90%, 91%, 92%, 93% of the amino acid sequence set forth in SEQ ID NO: Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity consist of or consist of. In some embodiments, the light chain of an anti-PCSK antibody comprises an amino acid sequence having one or more substitutions (eg, conservative substitutions), insertions or deletions as compared to an amino acid sequence selected from SEQ ID NO: 35, but comprising The light chain anti-PCSK9 antibody has the ability to bind PCSK9.

In a preferred embodiment, the substitution, insertion or deletion occurs in a region outside the CDR (e.g., in FR). Optionally, an anti-PCSK9 antibody of the invention comprises a post-translational modification to a light chain variable region, a heavy chain variable region, a light chain or a heavy chain.

In some embodiments, the substitution is a conservative substitution. A conservative substitution is one in which one amino acid is replaced by another amino acid in the same class, for example, one acidic amino acid is replaced by another acidic amino acid, one basic amino acid is replaced by another basic amino acid, or one neutral amino acid is passed through another neutral amino acid. Replacement. An exemplary substitution is shown in Table A below:

Table A

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Addition or deletion of a glycosylation site to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites. In some applications, modifications that remove unwanted glycosylation sites may be useful, or for example, removal of fucose modules to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277: 26733). In other applications, galactosidation modification can be performed to modify complement dependent cytotoxicity (CDC).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant to enhance, for example, antibody treatment involving abnormal angiogenesis and/or vascular permeability. The effectiveness of a disease or condition that is leaking. An Fc region variant can comprise a human Fc region sequence comprising an amino acid modification (eg, a substitution) at one or more amino acid positions (eg, a human IgGl, IgG2, IgG3, or IgG4 Fc region).

In certain embodiments, it may be desirable to produce a cysteine engineered antibody, such as a "thiocarba", wherein one or more residues of the antibody are replaced with a cysteine residue.

In certain embodiments, the antibodies provided herein can be further modified to contain other non-protein portions known in the art and readily available. Portions suitable for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene) Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof.

In some embodiments, the invention encompasses fragments of an anti-PCSK9 antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabody, linear antibody, single chain antibody molecule (eg, scFv); and multispecificity formed by antibody fragments Sexual antibodies. Papain digestion of antibodies produces two identical antigen-binding fragments, termed "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen binding sites and is still capable of cross-linking antigen.

In some embodiments, an anti-PCSK9 antibody of the invention is a humanized antibody. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in its entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633). Almagro & Fransson distinguish between rational approaches and empirical approaches. A rational approach is characterized by the generation of a few engineered antibody variants and assessing their binding or any other property of interest. If the design variant does not produce the expected results, then a new round of design and combined assessment is initiated. Rational approaches include CDR grafting, Resurfacing, Superhumanization, and Human String Content Optimization. In contrast, empirical approaches are based on generating large populations of human variants and using enrichment techniques or high-throughput screening to select the best clones. Thus, empirical approaches rely on reliable selection and/or screening systems that are capable of searching for a large number of antibody variants. In vitro display techniques, such as phage display and ribosome display, meet these requirements and are well known to the skilled artisan. Empirical approaches include FR libraries, Guided selection, Framework-shuffling, and Humaneering.

In some embodiments, an anti-PCSK9 antibody of the invention is a human antibody. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol 20: 450-459 (2008).

An antibody of the invention can be isolated by screening a combinatorial library for antibodies having the desired activity. For example, a variety of methods are known in the art for generating phage display libraries and screening for antibodies having the desired binding characteristics in these libraries. These methods are reviewed, for example, in Hoogenboom et al, Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Press, Totowa, NJ, 2001) and further, for example, in McCafferty et al, Nature 348:552-554. Clackso et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (edited by Lo, Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol 340(5): 1073-1093 (2004) Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284 (1-2): 119-132 (2004).

In some embodiments, the invention also encompasses anti-PCSK9 monoclonal antibodies ("immunoconjugates") conjugated to a therapeutic moiety, such as a cytotoxic agent or an immunosuppressive agent. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see for example WO05/103081. For example, cytotoxic agents include, but are not limited to, radioisotopes (eg, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , pb ²¹² and Lu radioisotopes) Chemotherapeutic agent or drug (eg, methotrexate, adriamicin, vinca alkaloids (vincristine), vinblastine, etoposide )), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents; growth Inhibitors; enzymes and fragments thereof such as nucleases; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antibiotics known Tumor or anticancer agent.

In some embodiments, an antibody of the invention may be monospecific, bispecific or multispecific. A multispecific monoclonal antibody can be specific for a different epitope of a target polypeptide or can contain an antigen binding domain that is specific for more than one target polypeptide. See, for example, Tutt et al. (1991) J. Immunol. 147: 60-69. The anti-PCSK9 mAb can be linked or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be functionally linked to one or more other molecules, such as another antibody or antibody fragment (eg, by chemical coupling, genetic fusion, non-covalent association, or otherwise) to produce a second or More bispecific or multispecific antibodies that bind specifically.

In some embodiments, an antibody of the invention binds to a human PCSK9 protein.

Nucleic acid of the present invention and host cell containing the same

In one aspect, the invention provides a nucleic acid encoding any of the above anti-PCSK9 antibodies or fragments thereof. The nucleic acid may encode an amino acid sequence comprising a light chain variable region and/or a heavy chain variable region of an antibody, or an amino acid sequence comprising a light chain and/or a heavy chain of an antibody.

In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector.

In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing a vector encoding an antibody include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, also to Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003). , pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of a yeast cell, a mammalian cell, or other cell suitable for use in the preparation of an antibody or antigen-binding fragment thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in partial or complete humans An antibody to the glycosylation pattern. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al, Nat. Biotech. 24: 210-215 (2006). Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for suspension growth can be used. Further examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney line (293 or 293 cells, eg eg Graham et al, J. Gen Virol. 36: 59 (1977) (described in) and so on. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 216 (1980)); and myeloma cell lines such as Y0. , NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for producing antibodies, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). .

In one embodiment, a method of making an anti-PCSK9 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, and optionally, under conditions suitable for expression of the antibody The antibody is recovered from the host cell (or host cell culture medium). For recombinant production of an anti-PCSK9 antibody, a nucleic acid encoding an antibody (such as the antibody described above) is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody).

Pharmaceutical composition and pharmaceutical preparation

The invention also encompasses compositions (including pharmaceutical or pharmaceutical formulations) comprising an anti-PCSK9 antibody and compositions comprising a polynucleotide encoding an anti-PCSK9 antibody. In certain embodiments, the composition comprises one or more antibodies that bind to PCSK9 or one or more polynucleotides that encode one or more antibodies that bind PCSK9. These compositions may also contain suitable pharmaceutical carriers such as those known in the art, including buffers.

Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk , glycerin, propylene, glycol, water, ethanol, etc. For the use of excipients and their use, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R. C. Rowe, P. J. Seskey and S. C. Owen, Pharmaceutical Press, London, Chicago. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, saccharin.

Preparation can be made by mixing an anti-PCSK9 antibody of the invention having the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th Ed., Osol, A., ed. (1980)). The pharmaceutical preparations of the anti-PCSK9 antibodies described herein are preferably in the form of a lyophilized preparation or an aqueous solution.

Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter including a histidine-acetate buffer.

The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient which is required for the particular indication being treated, preferably those which do not adversely affect each other's complementary activities. For example, it is desirable to also provide a statin. The active ingredient is suitably present in combination in an amount effective for the intended use.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing antibodies in the form of shaped articles such as films or microcapsules.

Antibody treatment and use

In one aspect, the invention relates to a method of inhibiting binding of PCSK9 to LDL-receptor (LDLR) in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies described herein or Its fragment. In another aspect, the invention relates to a method of lowering cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In one embodiment, the cholesterol is LDL-cholesterol. In another aspect, the invention relates to a method of lowering LDL-cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In some embodiments, the invention relates to a method of reducing serum LDL-cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In another aspect, the invention relates to a method of treating a condition associated with elevated LDL-cholesterol levels in a subject, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies described herein Or a fragment thereof. In one aspect, the invention relates to a method of treating a cholesterol-related disease, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In some embodiments, the invention relates to a method of treating hypercholesterolemia and/or hyperlipidemia, the method comprising administering to the subject an effective amount of any of the anti-PCSK9 antibodies or fragments thereof described herein. In one aspect, the invention relates to a method of treating any disease or condition that can be ameliorated, slowed, inhibited or prevented by eliminating, inhibiting or reducing PCSK9 activity. In some embodiments, a disease or condition that can be treated or prevented by the use of a statin can also be treated with any of the anti-PCSK9 antibodies or fragments thereof described herein. In some embodiments, a disease or condition that can benefit from preventing cholesterol synthesis or increased expression of LDLR can also be treated using any of the anti-PCSK9 antibodies or fragments thereof described herein. Preferably, the subject is a human.

In other aspects, the invention provides the use of an anti-PCSK9 antibody for the manufacture or preparation of a medicament for the treatment of a related disease or condition as mentioned above.

In certain embodiments, an antibody or antibody fragment of the invention against PCSK9 can be administered prophylactically to prevent or alleviate the onset of hypercholesterolemia, hyperlipidemia, cardiovascular disease, and/or any cholesterol-related disease. In certain embodiments, an antibody or antibody fragment of the invention against PCSK9 can be administered to treat existing hypercholesterolemia and/or hyperlipidemia. In some embodiments, an antibody or antibody fragment of the invention delays the onset of a condition and/or a condition associated with the condition.

In certain embodiments, the methods and uses described herein further comprise administering to the individual an effective amount of at least one additional therapeutic agent, eg, a statin, including but not limited to atorvastatin, fluvastatin , lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin or any combination thereof, for example,

or

In certain embodiments, the additional therapeutic agent is for preventing and/or treating atherosclerosis and/or cardiovascular disease. In certain embodiments, the additional therapeutic agent is used to reduce the risk of recurrent cardiovascular events. In certain embodiments, the additional therapeutic agent is for increasing HDL-cholesterol levels in a subject.

The above combination therapies include administering in combination (wherein two or more therapeutic agents are contained in the same or separate formulations) and separately, wherein administration of the anti-PCSK9 antibody of the invention may occur in additional therapeutic agents and/or Adjuvants are administered before, simultaneously and/or after.

The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if desired for topical treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. To some extent, depending on whether the administration is short-term or long-term, it can be administered by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various medication schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of a disease, a suitable dose of an antibody of the invention, when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease being treated, the type of antibody, the severity of the disease, and The process, whether the antibody is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in one treatment or through a series of treatments. Exemplary dosage ranges for the antibodies include 3-30 mg/kg.

Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-PCSK9 antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of PCSK9 in a biological sample. The term "detection" as used herein, includes quantitative or qualitative detection. In certain embodiments, the biological sample is a blood, serum or other liquid sample of biological origin. In certain embodiments, the biological sample comprises cells or tissues.

In one embodiment, an anti-PCSK9 antibody for use in a diagnostic or detection method is provided. In another aspect, a method of detecting the presence of PCSK9 in a biological sample is provided. In certain embodiments, the methods comprise detecting the presence of a PCSK9 protein in a biological sample. In certain embodiments, PCSK9 is human PCSK9. In certain embodiments, the method comprises contacting a biological sample with an anti-PCSK9 antibody as described herein under conditions that permit binding of the anti-PCSK9 antibody to PCSK9, and detecting whether a complex is formed between the anti-PCSK9 antibody and PCSK9. . The method can be an in vitro or in vivo method. In one embodiment, an anti-PCSK9 antibody is used to select a subject suitable for treatment with an anti-PCSK9 antibody, for example, wherein PCSK9 or LDL-cholesterol is a biomarker for selecting a patient.

In one embodiment, antibodies of the invention can be used to diagnose cholesterol-related diseases, such as hypercholesterolemia and/or hyperlipidemia.

In certain embodiments, a labeled anti-PCSK9 antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromophore labels, electron dense labels, chemiluminescent labels, and radioactive labels), as well as indirectly detected portions, such as enzymes or ligands, for example, By enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbrella Umbelliferone, Luciferiferase, for example, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydropyridazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lytic enzyme, carbohydrate oxidase, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, Heterocyclic oxidases such as uricase and xanthine oxidase, plus enzymes that utilize hydrogen peroxide to dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/affinity , spin labeling, phage labeling, stable free radicals, and more.

The invention is further illustrated by the following examples, which are to be understood by way of illustration and not limitation

Example

Example 1. Screening of antibodies against PCSK9 to determine the parent antibody

Antigen biotin labeling

The PCSK9 antigen (SEQ ID NO. 53) was biotinylated using the Pierce succinimidyl sulfonate biotin labeling kit according to the manufacturer's instructions. FITC-labeled goat anti-human immunoglobulin F(ab') kappa chain antibody (LC-FITC) was purchased from (Southern Biotech), polyethylene avidin (SA-PE) was purchased from (Sigma), streptavidin -633 (SA-633) was purchased from (Molec ular Probes). Streptavidin beads and a cell immunomagnetic bead separation column were purchased from Miltenyi LS.

Preliminary screening

Eight synthetic yeast-based antibody presentation libraries (from Adimab) were amplified according to existing methods (Xu et al., 2013; WO2009036379; WO 2010105256; W02012009568), each pool diversity reaching 1 ×10 ⁹ . Briefly, the first two rounds of screening were performed using magnetic activation cell sorting using Miltenyi's MACS system. First, each library of yeast cells (~1×10 ¹⁰ cells/library) was incubated in FACS wash buffer (phosphate buffer, containing 0.1% bovine serum albumin) for 15 minutes at room temperature, and the buffer contained the above. The prepared 100 nM biotinylated PCSK9 antigen was prepared. Wash once with 50 ml of pre-cooled FACS wash buffer, resuspend the cells with 40 ml of the same wash buffer, and add 500 μl of streptomycin microbeads for 15 minutes at 4 °C. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 5 ml of FACS wash buffer, and the cell solution was added to a Miltenyi LS column. After the addition was completed, the FACS wash buffer was washed 3 times for 3 ml each time. The Miltenyi LS column was removed from the magnetic field, eluted with 5 ml of growth medium, and the eluted yeast cells were collected and grown overnight at 37 °C.

The next round of sorting was performed using a flow cytometer: approximately 1 × 10 ⁸ yeast cells obtained by MACS screening were washed three times with FACS buffer for PCSK9 antigen containing low concentrations of biotin (100-1 nM). Alternatively, the PCSK9-Fc fusion antigen is cultured at room temperature. Discard the medium and wash the cells twice with FACS wash buffer, mix the cells with LC-FITC (1:100 dilution) and mix with SA-633 (1:500 dilution) or EA-PE (1:50 dilution) The reagents were mixed and incubated at 4 ° C for 15 minutes. The cells were eluted twice with pre-cooled FACS wash buffer and resuspended in 0.4 ml buffer and the cells were transferred to a separate tube with a filter. Cells were sorted using FACS ARIA (BD Biosciences).

This is followed by several rounds of screening to obtain competitive ligands and to remove non-specific conjugates (eg, membrane proteins of CHO cells). After the final rounds of sorting, the collected yeast cells were plated, cultured overnight at 37 ° C, and the target monoclonals were selected. The variable region of the obtained antibody was sequenced using the sanger method. A total of approximately 310 unique antibodies with variable region sequences were obtained and identified one by one.

These anti-PCSK9 antibody proteins were obtained by yeast expression and purification using Protein A affinity chromatography.

Antibody production purification

The yeast cells expressing the anti-PCSK9 antibody obtained by the screening were shaken at 30 ° C for 48 hours to express an antibody against PCSK9. After the end of the induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 min, and the supernatant was harvested. The anti-PCSK9 antibody in the supernatant was purified using Protein A, eluted with a pH 2.0 acetic acid solution, and the anti-PCSK9 antibody was harvested. The corresponding Fab fragment was obtained by papain digestion and purification with KappaSelect (GE Life Medical Group).

The gene DNA encoding the anti-PCSK9 antibody is obtained from the above-described yeast cell expressing the anti-PCSK9 antibody according to a conventional method in the art, and the gene DNA is cloned into a new expression vector (pCDNA3.1) according to a conventional method.

The above expression vector containing the antibody gene of interest and the transfection reagent Lipofectamine TM2000 (purchased from Invitrogen) were transiently transfected into cultured human kidney blast cell 293 cells according to the protocol provided by the manufacturer, the medium was discarded and the cells were treated with fresh medium. Dilute to 4X10 ⁶ /ml. The cells were cultured for 7 days at 37 ° C, 5% CO ₂ , and fresh medium was added every 48 hours. After 7 days, it was centrifuged at 13,000 rpm for 20 min. The supernatant was taken and the supernatant was purified with Protein A to give the antibody a purity of >95%.

ForteBio KD measurement (biofilm layer interferometry)

The ForteBio affinity assay was performed according to the existing method (Estep, P et al, High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p. 270-8). Briefly, the sensor was equilibrated in the assay buffer for 30 minutes, then on-line for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM of PCSK9 antigen for 5 minutes, after which the sensor was transferred to assay buffer for 5 minutes. The kinetic analysis was performed using a 1:1 binding model.

MSD-SET dynamics detection

The detection of equilibrium affinity is as described previously (Estep et al. 2013). Biotin-labeled PCSK9 antigen (b-PCSK9) obtained as described above was added to phosphate buffered saline (PBSF) containing 0.1% IgG-free BSA, and the anti-PCSK9Fab obtained as above was added to the final concentration of 10-100 pM. Or the mAb is serially diluted 3 to 5 times to obtain a Fab or mAb solution having a concentration of 5-100 nM. The antibody (diluted in 20 nM phosphate buffered saline) was coated on MSD-ECL plates at 4 ° C overnight or at room temperature for 30 minutes. 3% BSA was added and blocked at room temperature for 30 minutes at 700 rpm, followed by washing 3 times with washing buffer (PBSF + 0.05% Tween 20). The sample was added to the plate and placed in a shaker at room temperature for 700 seconds at 700 rpm for 1 second. 250 ng/ml sulfotag-labeled streptavidin (diluted in PBSF) was added and incubated for 3 minutes at room temperature. The buffer was washed 3 times and read using an MSD Sector Imager 2400 device to determine the antigen bound to the plate. The percentage of unbound antigen was obtained by titration of the antibody method, and it was found that the binding of the anti-PCSK9 Fab or mAb to the antigen was in accordance with the quadratic equation of metabolic kinetics.

Octet Red384 binding epitope identification

Binding epitope identification uses a standard sandwich-type interactive blocking assay. Target-specific control IgG was immobilized on the AHQ sensor and the Fc binding site over the sensor was blocked with an irrelevant human IgGl antibody. The sensor was placed in a 100 nM target antigen PCSK9 solution for 120 s and placed in a second 100 nM anti-PCSK9 antibody or ligand solution prepared as above. Data was read and processed by ForteBio Data Analysis Software 7.0 (from ForteBio). Binding of an antibody to a second antibody or ligand after binding to an antibody implies an unbound epitope (non-competitive), whereas no binding indicates epitope blocking (competitive or ligand blocking).

Through the above screening and identification work, we obtained antibodies that block the binding of PCSK9 and LDLR and bind to human mouse PCSK9. To obtain a higher affinity anti-PCSK9 antibody, we optimized the antibody ADI-02396 by the following method.

Example 2 Affinity Optimization of Anti-PCSK9 Antibody

VHmut screening

This method introduces mutations into the antibody heavy chain region by conventional mismatch PCR methods. During the PCR process, the base mismatch probability was increased to about 0.01 bp by using the 1 uM highly mutated base analogs dPTP and 8-oxo-dGTP.

The obtained mismatched PCR product was constructed by homologous recombination into a vector containing a heavy chain constant region. By this method, we obtained a secondary pool with a library capacity of 1x10 ⁷ under screening pressures including PCSK9 antigen titer, unlabeled antigen competition, and competition with parent antibody. Three rounds of successful screening were performed by the FACS method.

CDRH1/CDRH2 screening

The CDRH3 gene of the progeny antibody obtained by the VHmut method was constructed into a 1×10 ⁸ CDRH1/CDRH2 gene pool and subjected to 3 rounds of screening. In the first round, the MACS method was used, and in the second and third rounds, the FACS method was used, and the antibody antigen conjugate was subjected to affinity pressurization to select the antibody with the highest affinity.

First round of optimization: The first step was to increase the affinity of this human-mouse cross-active, ligand-competitive anti-PCSK9 antibody ADI-02396 (designated "parent" antibody). Briefly, a mutation is introduced into the parent antibody (using a "mismatch PCR" approach) to create a library of secondary yeast-based antibody presentations. A secondary pool of approximately 1 x ¹⁰⁷ size was eventually generated for enrichment of later higher affinity antibodies. Screening pressures include PCSK9 antigen titers, unlabeled antigen competition, and competition with parent antibodies. FACS technology is also used to screen target populations (see Chao et al. Nature Protocols, 2006 for specific methods of operation). After 2-3 rounds of enrichment, the obtained yeast is plated to obtain a monoclonal. After this work, a total of three improved affinity descendants, ADI-09111, ADI-09112 and ADI-09113, were obtained. Octet by measuring ForteBio, K _D range of these three antibodies are 1-10nM. Two progeny antibodies, ADI-09112 and ADI-09113, were used for affinity optimization in the second round.

A second round of affinity optimization: The second step was to increase the affinity of two human-rat cross-active, ligand-competitive anti-PCSK9 mAbs ADI-09112 and ADI-09113 (designated "parent" antibodies). In short, a secondary yeast-based antibody presentation library is created for each parent antibody. The CDR-H3 and light chain (LC) of the parent antibody were combined with the CDR-H1 and CDR-H2 of the gene in the existing yeast library (designated: "H1/H2" optimization). This ultimately resulted in 5 pools of approximately 1 x 10 ⁸ size for subsequent enrichment of higher affinity antibodies. The screening method is the same as the first round of screening. After 2-3 rounds of enrichment, yeast was applied to the plate to obtain a monoclonal. After this work, affinity-modified progeny antibodies were obtained, of which ADI-10085, ADI-10086 and ADI-10087 are variants of the CDR-H1 and CDR-H2 regions of ADI-09912, ADI-10088, ADI- 10089 and ADI-10090 are VH region variants of ADI-09113. See Table BD for relevant sequence information for the antibodies. The affinity of antibodies to human PCSK9 increased 10-fold, and the method by ForteBio MSD-SET tests showed that K _D in the range between 4-17pM ~ 200pM (Table 1, Table 2). Some antibodies have an affinity that is about 10 times higher than that of the control antibody. Identification of other antibody functions can further reduce the number of antibodies for preclinical development.

The sequence information and numbering of each anti-PCSK-9 antibody referred to in the present application are shown in Table B-D below:

Table B. Individual CDR Sequence Numbers for Exemplary Antibodies of the Present Application

Table C. Sequence numbering of each framework region of the heavy chain variable region and the light chain variable region of exemplary antibodies of the present application

Table D. Sequence numbering of the heavy chain variable region, light chain variable region, heavy chain and light chain sequences of exemplary antibodies of the present application

Table 1: Determination of antibody K _D values by ForteBio method

Table 2: MSD-SET Kinetic Determination of Antibody K _D Values

Example 3. Anti-PCSK-9 antibody inhibits binding of PCSK-9 to LDLR

The PCSK9 protein as described in Example 1 was diluted with PBS solution (phosphate buffer solution) to 400 nmol/L as a working solution. The anti-PCSK9 antibodies (ADI-10085, ADI-10086, ADI-10087, ADI-10088, ADI-10089, and ADI-10090) obtained in Example 2 were diluted with PBS solution to a concentration of 1000 nmol/L, 100 nmol/, respectively. L, 10 nmol/L, 1 nmol/L, and 0.1 nmol/L, solutions of various concentrations of control antibodies (Alirocumab, Evolocumab, Bococisumab, and Lodelcizumab) were prepared in the same manner. The PCSK9 working solution was mixed with the gradient-diluted individual anti-PCSK9 antibody samples or control samples in equal volumes. The LDLR-expressing CHO cells (CHO-LDLR) were resuspended in PBS solution and counted, and the cell concentration was adjusted to 4×10 ⁶ cells/ml with PBS solution, and seeded in a 96-well U-shaped cell culture plate to add 50 μl per well. The cell culture medium was mixed with 50 μl of a mixture of PCSK9 and anti-PCSK9 antibody, and the number of cells per well was 2.0 × 10 ⁵ , and the mixture was centrifuged at 200 g for 5 minutes at room temperature, and the supernatant was discarded. The final concentration of anti-His-FITC (R&D Systems) was diluted 1:200 with PBS solution to 2.5 μg/ml, and added to a 96-well plate at 100 μl per well for 30 minutes in an ice bath. Centrifuge at 200 g for 5 minutes at room temperature, gently discard the supernatant, add 150 μl of PBS solution to each well, centrifuge at 200 g for 5 minutes at room temperature, and gently discard the supernatant for 3 times. 80 μl/well of PBS solution was added to each experimental well, and the cells were resuspended by pipetting several times. Cellular fluorescence signal values were measured by flow cytometry.

For the fluorescent signal detected by the experiment, please refer to Table 3 below.

Table 3: Fluorescence signal values

The raw data of Table 3 was analyzed by GraphPad Prism6 software to obtain Figure 1.

The experimental results show that the anti-PCSK9 antibody has a comparable ability to block the binding of PCSK9 to LDLR compared to the control antibody.

Example 4 Cell LDL-c Uptake Analysis Experiment

Remove the HepG2 cell cryopreservation tube from the liquid nitrogen storage tank, melt in a 37 °C rapid water bath, transfer the cell suspension into a 15 ml centrifuge tube, and slowly add 4 ml of room temperature growth medium (90% DMEM + 10% FBS, of which DMEM and FBS are purchased). After centrifugation at 1000 r/min for 1 minute at room temperature, the pellet was resuspended in fresh growth medium, transferred to a culture flask and cultured at 37 ° C, 5% CO ₂ . After the logarithmic growth phase of HepG2 cells was washed twice with PBS solution, 1 ml of 0.25% trypsin (purchased from Gibco) was added for 3 minutes, and 6 ml of growth medium was added to resuspend the cells to terminate the reaction. HepG2 cells were adjusted to 0.8 × 10 ⁶ /ml with growth medium, and seeded in a black bottom transparent poly-D-lysine-coated 96-well cell culture plate (purchased from Nunc) at 100 μL per well, 37 ° C Incubate for 6-7 hours in a 5% CO ₂ incubator. The growth medium was discarded, replaced with assay medium (DMEM + 5% FBS), 100 μl/well, and cultured overnight at 37 ° C in a 5% CO ₂ incubator. Antibody samples (ADI-10085, ADI-10087, ADI-10088, ADI-10089) were diluted to 66.7 nmol/L with assay medium, respectively. The 66.7 nmol/L sample was used as the starting concentration, and a 4-fold gradient dilution was performed. The positive control antibodies (Alirocumab, Evolocumab, and Lodelcizumab) and the negative controls (LDL, PCSK9 + LDL, and IgG) were subjected to the same operation. Each of the obtained concentration gradient samples was separately mixed with 60 μl and 60 μl of 66.7 nmol/L of PCSK9 in equal volumes to obtain each mixed solution. The blank control was 120 μl of assay medium. The 96-well plate was aspirated, 50 μl of the above mixture and a blank control sample were added to each well, and incubated at 37 ° C in a 5% CO ₂ incubator for 1 hour. The plate was taken out, 50 μl of each well was added, and 6 μg/ml of BODIPY-labeled LDL solution (purchased from life technologies) diluted with an analysis medium was used, and the plate was incubated at 37 ° C, 5% CO ₂ for 4 hours. The medium was discarded, and the plate was washed with 100 μl of PBS solution per well and discarded. Wash twice and add 100 μl of PBS solution to each well. Fluorescence values were read using a Spectra Max I3 plate reader.

The obtained fluorescence value raw data are listed in Table 4 below, and the data disclosed in Table 4 was analyzed by GraphPad Prism6 software to obtain Fig. 2. From the experimental results, it was found that the anti-PCSK9 antibody (ADI-10085, ADI-10087, ADI-10088, ADI-10089) enhanced the fluorescence value by about two times in the case of HepG2 cells compared to the fluorescence value of the anti-PCSK9 antibody. These data demonstrate that each of the anti-PCSK9 antibodies disclosed in the present application can increase the resilience of LDLR in HepG2 cells, inhibit the decrease in LDK-c uptake induced by PCSK9, thereby increasing the uptake of LDL-c by HepG2 cells, and at 16.8 The gradients of nM and 66.7 nM were superior to positive control antibodies.

Table 4: Fluorescence signal values

Example 5. Cellular LDLR internalization analysis experiment

PCSK9 can directly bind to LDLR to promote LDLR internalization, enter hepatocytes and transport to lysosomal degradation, thereby reducing LDLR expressed on the cell surface and increasing the level of LDL-c in serum. Anti-PCSK9 antibody blocks the binding of PCSK9 to LDLR, thereby reducing the ability of PCSK9 to consume LDLR. In this experiment, CHO-LDLR cells were incubated with anti-PCSK9 antibody and PCSK9 protein solution, and the fluorescence value of LDLR was detected by flow cytometry. The anti-PCSK9 antibody and the positive control antibody (Evolocumab) fluorescence values were compared to determine the biological activity of the anti-PCSK9 antibody on cellular LDLR internalization.

The above PCSK9 protein was configured to a concentration of 50 μg/ml using RPMI 1640 cell culture medium (Gibco), 60 μl, 1000 nm anti-PCSK9 antibody (ADI-10085 and ADI-10087) was mixed with PCSK9 (50 μg/ml) solution, and then uniformly incubated. The positive antibody control was treated identically for 30 minutes. CHO cells and CHO-LDLR cells were centrifuged separately, centrifuged at 500 g for 3 minutes at room temperature, resuspended in PBS solution and adjusted to a cell density of 2×10 ⁶ /ml, and added to a 96-well U-shaped plate at 100 μl/well. 100 μl/well of the mixed sample was added to the culture plate in quadruplicate, blown evenly, and incubated at 4 ° C for 4 hours. Thereafter, it was washed three times with 200 μl of a PBS solution, and centrifuged at 500 g for 3 minutes at room temperature. 5 μl of anti-LDLR-PE (Beijing Yiqiao Company, Cat. No. 20131-R301-P) was added to 100 μl of PBS solution for dilution, and then 100 μl per well was added to a 96-well U-shaped plate and incubated for 30 minutes in the dark. The cells were washed three times with 200 μl of PBS solution, centrifuged at 500 g for 3 minutes, and the obtained cells were resuspended in cell culture medium, and the fluorescence signal of LDLR protein on the surface of PE labeled with CHO-LDLR cells was detected by flow cytometry. See Table 5. The raw data of Table 5 was analyzed by GraphPad Prism6 software to obtain Figure 3.

As can be seen from the results of Table 5, the antibodies obtained in the present application effectively prevented LDLR internalization of cells.

Table 5: Fluorescence signal values

Example 6: Epitope recognition of anti-PCSK antibodies of the invention

Prior to characterization, the anti-PCSK antibody of the invention (using ADI-10087 in this example, using an Ultrafelx III MALDI ToF ToF mass spectrometer (Bruker) equipped with a CovalX's HM4 interaction module (CovalX AG, Zurich, Switzerland) The integrity and aggregation levels of human PCSK-9 as antigens were determined separately (experimental methods and results analysis are as follows), and non-covalent between anti-PCSK antibodies and human PCSK9 were not detected. polymerization. Human PCSK9 (hereinafter referred to as PCSK9-WT) was formed by non-covalent bonding of two subunits of 59.983 kDa (hereinafter referred to as PCSK9-WT Reduced) and 13.749 kDa.

A. Characterization of antibody/antigen complexes

1. Materials and methods

1.1 Instrument

To characterize the complex, the molecular weight was measured using an Ultraflex III MALDI ToF ToF mass spectrometer (Bruker) equipped with a CovalX HM4 interaction module.

CovalX's interaction module includes a specialized detection system designed to optimize detection with molecular weights up to 2MDa with nanomolar sensitivity.

1.2 Sample preparation:

Control experiment

Antibody/antigen complexes were prepared at the following concentrations:

1 μl of the obtained antibody/antigen mixture was mixed with 1 μl of a sinapic acid substrate supersaturated solution (10 mg/ml, acetonitrile/water (1:1, v/v), TFA 0.1%, K200 MALDI Kit), and then 1 μl was taken at MALDI Board (SCOUT 384, AchorChip). After crystallization at room temperature, the plates were introduced into a MALDI ToF mass spectrometer and analyzed immediately. The analysis was repeated three times.

Cross-linking experiment

The mixture prepared for the control experiment (the remaining 9 μl) was cross-linked using the K200 MALDI MS Assay Kit of CovalX. 9 μl of the antibody/antigen mixture was mixed with 1 μl of K200 Stabilizer reagent (2 mg/ml, CovalX AG, Zurich, Switzerland) and incubated at room temperature for 180 minutes, after which samples were prepared for MALDI analysis as described in the control experiments.

1.3High-Mass MALDI MS Analysis

MALDI ToF MS analysis was performed using CovalX's HM4 interaction module with a standard nitrogen laser ion source and can be focused on different mass ranges from 0-2000 kDa.

The parameter used is

Mass spectrometer:

Linear and positive ion mode

Ion source 1: 20kV

Ion source 2: 17kV

Lens: 12kV

Pulse ion extraction: 400ns

HM4:

Gain voltage: 3.14kV

Acceleration voltage: 20kV

The mass spectrometer was subjected to external mass calibration using a mixture of proteins containing Insulin, BSA and IgG prior to use. For each sample, 3 points were analyzed (300 laser shots per point). The spectrum presented corresponds to the sum of 300 laser illuminations. MS data was analyzed using CovalX's Complex Tracker Analysis Software version 2.0.

2. Results

2.1 anti-PCSK9 antibody / PCSK9-WT

2.1.1 Interaction Analysis

Control experiment

In the control experiment, both the antigen PCSK9-WT Reduced and the anti-PCSK9 antibody were detected, and the detected molecular weights were MH+=59.716 kDa and MH+=146.769 kDa, respectively (Fig. 6, control).

观察到的分子量(kDa)Observed molecular weight (kDa)
59.71659.716	PCSK9-WT ReducedPCSK9-WT Reduced
146.769146.769	抗PCSK9抗体anti-PCSK9 antibody

Cross-linking experiment

In the cross-linking experiment, the antibody/antigen complex was incubated with the cross-linking agent K200 for 180 minutes and the molecular weight was measured with MALDI ToF. After cross-linking, in addition to the two peaks detected in the control experiment, four additional peaks were detected: MH+=214.902 kDa, MH+=229.111 kDa, MH+=276.105 kDa and MH+=290.815 kDa (Fig. 6, cross-linking) ).

Using the Complex Tracker software, we overlayed the control and cross-linking spectra and resolved four non-covalent complexes with the following composition (Figure 6, overlap):

3. Conclusion of antibody/antigen complex characterization

Characterization of the antibody/antigen complex revealed that the antigen PCSK9-WT Reduced, PCSK9-WT can bind to the anti-PCSK9 antibody.

B. Characterization of antibody/antigen complex molecular interface (Molecular Interface)

For high-resolution determination of the binding epitope of the anti-PCSK9 antibody and the antigen PCSK9-WT, the antibody/antigen complex was incubated with the cross-linking agent DSS d0/d12, followed by trypsin (Trypsin) and chymotrypsin ( Chymotrypsin), aspartate N-terminal endonuclease (Asp-N), elastase (Elastase) and thermolysin (Thermolysin) are enzymatically hydrolyzed, and the cross-linked peptide obtained after enzymatic hydrolysis passes through the nanoliter liquid phase. Chromatography and high resolution mass spectrometry in-line tandem systems (nLC-LTQ-Orbitrap MS/MS) were identified and analyzed using XQuest and Stavrox software.

The antigen PCSK9-WT alone (without anti-PCSK9 antibody) was cross-linked and sequence characterized (experimental steps were cross-linked and analyzed with the antibody/antigen complexes listed below). Based on the results of enzymatic digestion and mass spectrometry analysis, the sequence coverage of PCSK9-WT was 88.77%. The amino acid sequence and the peptides identified by different endonuclease digestions are shown in Figure 7. . The experimental results of these peptides and antibody/antigen complexes were integrated and analyzed to improve the accuracy of epitope identification.

1. Materials and methods

1.1 Instrument

The Ultimate 3000 (Dionex) nanoliter LC system is connected in series with the LTQ-Orbitrap XL mass spectrometer (Thermo Scientific).

1.2 Sample preparation:

Antibody/antigen complex

To obtain an antibody/antigen mixture at a final concentration of 0.5 μM / 4 μM, 5 μl of antibody (concentration 1 μM) was mixed with 5 μl of antigen sample (concentration 8 μM). The mixture was incubated at 37 ° C for 180 minutes.

Cross-linking reaction

1 mg of DSS (d0) crosslinker (Thermo Scientific) was mixed with 1 mg of deuterated DSS (d12) crosslinker (CovalX AG), and 1 ml of DMF was added to obtain a 2 mg/ml DSS d0/d12 solution. 10 μl of the previously prepared antibody/antigen mixture was mixed with 1 μl of the prepared cross-linking agent DSS d0/d12 solution (2 mg/ml), and incubated at room temperature for 180 minutes for crosslinking.

Reduction/alkylation

10 μl of the previously prepared cross-linked antibody/antigen complex was mixed with 40 μl of ammonium hydrogencarbonate (25 mM, pH 8.3), 2 μl of DTT (500 mM) was added, and incubated at 55 ° C for 1 hour. After the incubation, 2 μl of iodoacetamide (1 M) was added and incubated for 1 hour at room temperature in a dark room. After the incubation, 120 μl of proteolytic buffer (owned by each endonuclease product) was added.

Trypsin enzymatic hydrolysis

145 μl of the reduced/alkylated cross-linked antibody/antigen complex was mixed with 0.70 μl of Trypsin (Roche Diagnostic) in a ratio of 100:1 (protein: enzyme, w/w) and incubated overnight at 37 °C.

Chymotrypsin enzymatic hydrolysis

145 μl of the reduced/alkylated cross-linked antibody/antigen complex was mixed with 0.35 μl of Chymotrypsin (Roche Diagnostic) in a ratio of 200:1 (protein: enzyme, w/w), and incubated overnight at 25 °C.

Asp-N proteolysis

145 μl of the reduced/alkylated cross-linked antibody/antigen complex was mixed with 0.35 μl of Asp-N (Roche Diagnostic) in a ratio of 200:1 (protein: enzyme, w/w) and incubated overnight at 37 °C.

Elastase enzymatic hydrolysis

145 μl of the reduced/alkylated cross-linked antibody/antigen complex was mixed with 0.70 μl of Elastase (Roche Diagnostic) in a ratio of 100:1 (protein: enzyme, w/w) and incubated overnight at 37 °C.

Thermolysin enzymatic hydrolysis

145 μl of the reduced/alkylated cross-linked antibody/antigen complex was mixed with 1.40 μl of Thermolysin (Roche Diagnostic) in a ratio of 50:1 (protein: enzyme, w/w) and incubated overnight at 70 °C.

After overnight hydrolysis, the reaction was stopped by the addition of 1% formic acid.

nLC-LTQ-Orbitrap MS/MS Analysis

10 μl of the digested peptide solution was injected into a nanoliter liquid chromatography system (Ultimate 3000, Dionex), and peptide identification was performed by in-line LTQ-Orbitrap XL (Thermo Scientific). The liquid chromatography and mass spectrometry operating parameters are:

-流动相A- Mobile phase A	-95/05/0.1水/乙腈/甲酸v/v/v-95/05/0.1 water/acetonitrile/formic acid v/v/v
-流动相B- Mobile phase B	20/80/0.1水/乙腈/甲酸v/v/v20/80/0.1 water/acetonitrile/formic acid v/v/v
-洗脱梯度- elution gradient	在35分钟内5-40％B5-40% B in 35 minutes
-进样体积- injection volume	10μl10μl
-预柱(pre-column)-Pre-column	300μm ID x 5mm C4 PepMap ^TM 300μm ID x 5mm C4 PepMap ^TM

-预柱流速- Pre-column flow rate	30μl/min30μl/min
-分析柱(analytical column)-analytical column	75μm ID×5cm C4 PepMap ^TM 75μm ID×5cm C4 PepMap ^TM
-分析柱流速- Analyze column flow rate	200nl/min200nl/min

-Needle voltage-Needle voltage	1.8kV1.8kV
-Capillary voltage-Capillary voltage	5kV5kV
-μscan MS-μscan MS	11
-μscan MS2-μscan MS2	11
-MS range m/z-MS range m/z	300-1700300-1700
-MS/MS strategy-MS/MS strategy	MS+6 CID MS/MSMS+6 CID MS/MS
-Min.Signal required-Min.Signal required	500500
-Ion isolation window-Ion isolation window	3m/z units3m/z units
-Normamized collision energy-Normamized collision energy	35％35%
-Default charge state-Default charge state	22
-Activation q-Activation q	0.250.25
-Activation time-Activation time	3030
-Dynamic exclusion-Dynamic exclusion	ONON
-Dynamic exclusion params-Dynamic exclusion params	RC 1,RD 30s,ED 30s RC 1, RD 30s, ED 30s
-Charge state screening-Charge state screening	ONON
-Charge state rejection-Charge state rejection	ONON
-Charge state rejection Params-Charge state rejection Params	+1and Unassigned Rejected+1and Unassigned Rejected

data analysis

Cross-linked peptides were analyzed using Xquest version 2.0 and Stavrox 2.1 software.

2. Results

2.1.1 Trypsin enzymatic hydrolysis

After the DSS d0/d12 cross-linked antibody/antigen complex was digested with Trypsin, a cross-linked peptide between the anti-PCSK9 antibody and the antigen PCSK9 was detected by nLC-LTQ-Orbitrap MS/MS analysis. This cross-linked peptide was detected by both Xquest and Stavrox software.

2.1.2Chymotrypsin enzymatic hydrolysis

The DSS d0/d12 cross-linked antibody/antigen complex was digested with Chymotrypsin, and n cross-linking peptides between the anti-PCSK9 antibody and the antigen PCSK9 were detected by nLC-LTQ-Orbitrap MS/MS analysis. These cross-linked peptides were detected by both Xquest and Stavrox software.

2.1.3Asp-N proteolysis

After the DSS d0/d12 cross-linked antibody/antigen complex was digested with Asp-N, nC-LTQ-Orbitrap MS/MS analysis did not detect any cross-linked peptide between the anti-PCSK9 antibody and the antigen PCSK9.

2.1.4Elastase enzymatic hydrolysis

After the DSS d0/d12 cross-linked antibody/antigen complex was digested with Elastase, nC-LTQ-Orbitrap MS/MS analysis did not detect any cross-linked peptide between the anti-PCSK9 antibody and the antigen PCSK9.

2.1.5 Thermolysin enzymatic hydrolysis

After the DSS d0/d12 cross-linked antibody/antigen complex was digested with Thermolysin, nCl-LTQ-Orbitrap MS/MS analysis did not detect any cross-linking peptide between the anti-PCSK9 antibody and the antigen PCSK9.

3. Conclusion

By chemical cross-linking and nLC-LTQ-Orbitrap MS/MS analysis, we were able to characterize the interaction interface between the anti-PCSK9 antibody and the antigen PSCK9.

Our analysis indicated that the epitope of this monoclonal antibody contains a region formed by several PSCK9 amino acid sites: 48 (tyrosine) (corresponding to Y78 of human PCSK9 shown in SEQ ID NO: 53), 56 (threonine) (corresponding to T86 of human PCSK9 shown in SEQ ID NO: 53), 57 (histidine) (corresponding to H87 of human PCSK9 shown in SEQ ID NO: 53) and 74 (arginine) (corresponding to R104 of human PCSK9 shown in SEQ ID NO: 53). The result is shown in Figure 8.

A site that binds to the antigen PSCK9 on an anti-PCSK9 antibody, including 24 arginine on the antibody light chain (complementarity determining region 1), 26 serine (complementarity determining region 1), and 28 serine (complementarity determining region 1) And 96 phenylalanine (complementarity determining region 2), and 60 tyrosine (complementarity determining region 2) and 102 serine (complementarity determining region 3) on the heavy chain.

Example 7 Anti-PCSK9 Antibody Reduces Blood Lipid Effect in Healthy SD Rats

The test antibody (anti-PCSK9 antibody ADI-10087) was administered to SPF grade SD rats according to conventional methods in the art, wherein the female rats weighed about 254-294 g, about 9-12 weeks old; the male rats weighed about 369-420 g. , about 9 to 12 weeks old. Each group was administered in a single dose, and the dosage regimen is shown in Table 6.

Table 6: Dosage design and administration

The animals in each group were collected according to the conventional method at the following time points: 0h before administration (D1), 72h after administration (D4), 168h (D8), 336h (D15), 504h (D22), 672h (D29), 840h (D36). The cells were collected into a test tube without an anticoagulant, and placed on ice for 2 to 8 ° C, centrifuged at 5000 rpm / min for 10 minutes, serum was collected, and LDL-C and HDL-C were measured by a Hitachi-7060 automatic biochemical analyzer. Based on the blood lipid analysis data, the relative pre-dose (baseline) rate of change (%LDL-C and %HDL-C) of LDL-C and HDL-C at each time point was calculated. According to the experimental results, it was found that a single subcutaneous administration of 3 to 30 mg/kg of the anti-PCSK9 antibody of the present invention can reduce serum LDL-C and HDL-C levels in a dose-dependent manner (Fig. 9 and Fig. 10). For example, there was a significant decrease from the baseline level at 3 days, 7 days, 14 days, and 21 days after administration, respectively. In addition, Applicants also found that serum LDL-C and HDL-C levels were not significantly reduced after a single subcutaneous administration of 10 mg/kg Evolocumab in rats.

The above method can also be carried out by the same method for the other antibodies of the present invention.

Example 8 Anti-PCSK9 antibody against hypolipidemic effect in healthy cynomolgus monkeys

The test antibody (anti-PCSK9 antibody ADI-10087) is administered to cynomolgus monkeys according to conventional methods in the art, wherein the female animal weighs about 2-4 kg, the age is about 3-5 years old, the male animal weighs about 3-5 kg, and the age is about 3 - 5 years old. See Table 7 for the dosing schedule, in which groups 1 to 5 are single administrations, and group 6 is administered once a week for a total of 4 administrations.

Table 7: Dosage design and administration

For groups 1 to 5, blood was collected from the contralateral forelimb or hind limb subcutaneous vein or inguinal femoral artery/inguinal vein of the animal's administration limb at the following time points: 0 h before administration and 24 h (D2), 72 h after administration. (D4), 120h (D6), 168h (D8), 336h (D15), 504h (D22), 672h (D29), 840h (D36), 1008h (D43), 1176h (D50), and 1344h (D57). For group 6, blood was collected according to the above method at the following time points: 0 h before the first administration and 24 h after the administration (D2), 72 h (D4), 120 h (D6), 168 h (D8, before the second administration), 336 h (D15, before the third administration) Blood was taken. The last administration was 0 h before administration, 24 h (D2), 72 h (D4), 120 h (D6), 168 h (D8), 336 h (D15), 504 h (D22), 672 h (D29), 840 h (12 h after the start of administration). Blood was collected from D36), 1008h (D43), 1176h (D50), and 1344h (D57).

Collect whole blood into the test tube containing the coagulant and the separation gel, place it on the ice and centrifuge at 2-8 ° C, centrifuge at 5000 rpm/min, and centrifuge for 10 min. After the whole blood sample is collected, total cholesterol (TC), LDL-C, Determination of HDL-C. Based on the blood lipid analysis data, the rate of change of LDL-C and HDL-C relative to the pre-dose (baseline) at each time point (%LDL-C and %HDL-C) was calculated. According to the experimental results, it was found that the serum LDL-C (Fig. 11) and TC (Fig. 13) levels were significantly decreased after a single subcutaneous administration of 3, 10, 30 mg/kg of the anti-PCSK9 antibody (ADI-10087) of the cynomolgus monkey. And showed a more obvious dose-effect relationship, significantly lower than the baseline level 3 to 28 days after administration. It is shown that the antibodies disclosed herein can be effectively used to reduce conditions and/or conditions associated with LDL-C and TC, for example, to lower blood lipids.

Anti-PCSK9 antibody administration had no significant effect on serum HDL-C levels in cynomolgus monkeys (Fig. 12).

However, the Applicant has surprisingly found that cynomolgus monkeys were administered subcutaneously 10 mg/kg of the anti-PCSK9 antibody and Evolocumab of the present application, respectively, and found that the time period during which LDL-C showed a significant decrease from the baseline level after Evolocumab administration was only 14 days. Shorter than the same dose of the antibody of the present application for 21 days after administration, the anti-PCSK9 antibody of the present application significantly reduced LDL-C for longer than Evolocumab.

Claims

An anti-PCSK9 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein said HCVR comprises a complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3 and said LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises at least 90%, 91%, 92%, 93% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 7, 8, 9, 10, 11, 12, 13 and , or consisting of, or consisting of, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity, HCDR2 comprising and selected from the group consisting of SEQ ID NOs: 2, 14, 15, 16 Amino acid sequences of 17 and 21 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity or consisting of Composition, and HCDR3 comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 18, 19 and Or consisting of or consisting of an amino acid sequence of 99% identity or 100% identity; wherein LCDR1 comprises at least 90%, 91%, 92 of the amino acid sequence of SEQ ID NO: % or 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical amino acid sequence consisting of, and the LCDR2 comprises at least the amino acid sequence of SEQ ID NO: 5 Amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity, or consists of LCDR3 comprising SEQ ID NO The amino acid sequence shown in 6 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity or Its composition.
The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 25, 26, 27, 28, 29, 30, 31, 32 and 33 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity consists of or consists of.
The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the light chain variable region LCVR comprises at least 90%, 91%, 92%, 93%, 94%, 95 of the amino acid sequence of SEQ ID NO: Amino acid sequences of %, 96%, 97%, 98% or 99% identity or 100% identity consist of or consist of.
The antibody or antigen-binding fragment thereof of claim 1 comprising a heavy chain, wherein said heavy chain comprises and selected from the group consisting of SEQ ID NOs: 34, 36, 37, 38, 39, 40, 41, 42, 43, and 44 The amino acid sequence has or consists of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity.
The antibody or antigen-binding fragment thereof of claim 1 or 4, comprising a light chain, wherein said light chain comprises at least 90%, 91%, 92%, 93%, 94 of the amino acid sequence of SEQ ID NO: Amino acid sequences of %, 95%, 96%, 97%, 98% or 99% identity or 100% identity consist of or consist of.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a humanized antibody or a human antibody.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is a fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv or (Fab') 2 fragments.
The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, comprising a framework sequence, wherein at least a portion of the framework sequence is a human consensus framework sequence.
An isolated nucleic acid encoding the anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.
A vector comprising the nucleic acid of claim 10, preferably the vector is an expression vector.
A host cell comprising the vector of claim 11, preferably the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of a yeast cell, a mammalian cell or other cell suitable for the preparation of an antibody or antigen-binding fragment thereof.
A method of producing an anti-PCSK9 antibody or antigen-binding fragment thereof, the method comprising cultivating the host of claim 12 under conditions suitable for expressing a nucleic acid encoding the anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. The cell, optionally isolating the antibody or antigen-binding fragment thereof, optionally the method further comprising recovering the anti-PCSK9 antibody or antigen-binding fragment thereof from the host cell.
A pharmaceutical composition comprising the anti-PCSK9 antibody or antigen-binding fragment thereof of any one of claims 1 to 9, and optionally a pharmaceutically acceptable carrier.
A method of lowering a cholesterol level of a subject, the method comprising administering to the subject an effective amount of the anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, preferably, the cholesterol is LDL-cholesterol is more preferably serum LDL-cholesterol.
A method of treating a cholesterol-related disease in a subject, the method comprising administering to the subject an effective amount of the anti-PCSK9 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, preferably the cholesterol-related The disease is hypercholesterolemia and/or hyperlipidemia.
The method of claim 15 or 16, further comprising administering to the subject an effective amount of a second drug, wherein the anti-PCSK9 antibody or antigen-binding fragment thereof is a first drug, preferably the second The medicament comprises a statin, more preferably the statin is selected from the group consisting of atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, roche Ruvastatin, simvastatin, and any combination thereof.
A method of inhibiting binding of PCSK9 to LDLR in a subject, the method comprising administering to the subject an effective amount of the anti-PCSK9 antibody or antigen-binding fragment thereof of any one of claims 1 to 9.
A method of detecting a PCSK9 protein in a sample, the method comprising

(a) contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-9;

(b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and the PCSK9 protein.
An anti-PCSK9 antibody or antigen-binding fragment thereof that binds to an epitope of PCSK9 comprising one or more of residues Y78, T86, H87 and R104 of human PCSK9 amino acid sequence SEQ ID NO:53.
An anti-PCSK9 antibody or antigen-binding fragment thereof that binds to the same epitope as any of the antibodies of claim 1 or antigen-binding fragments thereof.